This invention relates to a water-soluble tetraazaporphin, a fluorochrome for labeling, a substance derived from an organism which has been labeled with the fluorochrome for labeling, a reagent comprising any of them, and a fluorescence analysis process using any of them.
Since early times, label methods have been utilized for investigation of various substances such as molecules, cells, antigens, antibodies, DNA, RNA, polypeptides, etc. A label method using a radioisotope (RI) has heretofore been widely utilized because it has been studied for a long period of time. Since RI entails a severe exposure hazard, employment of RI requires both a special license and a special laboratory, and has been possible only for specified persons in specified facilities.
On the other hand, a method using a coloring substance, chemiluminescence method, fluorescence method, etc. are noted as label methods free from danger because they need not use RI. The method using a coloring substance cannot have a high detection sensitivity and hence is not so useful as to replace the RI method thereby. On the other hand, the chemiluminescence method and the fluorescence method are considered safe label methods which can replace the RI method, because their detection sensitivity can be enhanced. However, in the chemiluminescence method, luminescence is caused by combination of two or more chemical reactions, so that a complicated procedure is required. Therefore, fluorescence label method is the most excellent label method from the viewpoint Of safety, simplicity and high sensitivity.
There have been known only dyes which emit fluorescence in the ultraviolet region, but Rhodamine dyes and oxazine dyes have recently come to be known to the art as dyes which can be excited by means of an argon laser or a He--Ne laser.
As a light source, a compact semiconductor laser (670 to 840 nm) has recently become available at a low price. It is likely to become a leading light source because of the demand for an inexpensive, small and light instrument. However, there is a problem in that conventional Rhodamine dyes, oxazine dyes and the like cannot be used when the semiconductor laser is used.
It has recently been proposed that phthalocyanine capable of showing a proper fluorescence quantum yield and a high water-solubility is used as a fluorochrome for labeling (International Publication Nos. WO 88/04777 and WO 90/02747). However, the absorption maximum wavelength region and fluorescence emission wavelength region in Q-band of phthalocyanine derivatives are in a range of 670 to 690 nm, so that the phthalocyanine derivatives cannot be excited by means of a semiconductor laser emitting a wavelength of 700 nm or more. Moreover, when a semiconductor laser emitting a wavelength of 670 to 680 nm is used, its emitting wavelength region and the fluorescence emission wavelength region-overlap each other, so that it is impossible to judge whether light detected is derived from fluorescent radiation emitted or laser beam scattered. Therefore, the phthalocyanine derivatives cannot be utilized. In short, the phthalocyanine derivatives have a fatal defect in that they cannot be used at all in a system using a semiconductor laser which is a leading system.
Furthermore, in a system in which a substance in a living body, for example, a heme in blood is present together with an analyte to be measured, measurement of the analyte is restricted by the substance in the living body because the heme has an absorption wavelength region of 700 nm or less which overlaps with that of phthalocyanine.